Principles of Virology. Jane Flint

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      In most cases, the 50% end point does not fall on a dilution tested as shown in the example; for this reason, various statistical procedures have been developed to calculate the end point of the titration. In one popular method, the dilution containing the ID₅₀ is identified by interpolation between the dilutions on either side of this value. The assumption is made that the location of the 50% end point varies linearly with the log of the dilution. Because the number of test units used at each dilution is usually small, the accuracy of this method is relatively low. For example, if six test units are used at each 10-fold dilution, differences in virus titer of only 50-fold or more can be detected reliably. The method is illustrated in the second example below, in which the lethality of poliovirus in mice is the end point. Eight mice were inoculated per dilution. In the method of Reed and Muench, the results are pooled, as shown in the table below, which equalizes chance variations (another way to achieve the same result would be to utilize greater numbers of animals at each dilution). The interpolated value of the 50% end point, which in this case falls between the 5th and 6th dilutions, is calculated to be 10^–6.5. The virus sample therefore contains 10^6.5 LD₅₀ (50% lethal dose). The LD₅₀ may also be calculated as the concentration of the stock virus in PFU per milliliter (1 × 10⁹) times the 50% end-point titer. In the example shown, the LD₅₀ is 3 × 10² PFU.

       Reed LJ, Muench H. 1938. A simple method of estimating fifty percent endpoints. Am J Hyg 27:493–497.

Dilution	Alive	Dead	Total alive	Total dead	Mortality ratio	Mortality (%)
10–2	0	8	0	40	0/40	100
10–3	0	8	0	32	0/32	100
10–4	1	7	1	24	1/25	96
10–5	0	8	1	17	1/18	94
10–6	2	6	3	9	3/12	75
10–7	5	3	8	3	8/11	27

      Although the linear nature of the dose-response curve indicates that a single particle is capable of initiating an infection (one-hit kinetics) (Fig. 2.8), the high particle-to-PFU ratio of many viruses demonstrates that not all virus particles are successful. High values are sometimes caused by the presence of noninfectious particles with genomes that harbor lethal mutations or that have been damaged during growth or purification (defective particles). An alternative explanation is that although all viruses in a preparation are in fact capable of initiating infection, not all of them succeed because of the complexity of the infectious cycle. Failure at any one step in the cycle prevents completion. In this case, a high particle-to-PFU ratio indicates not that most particles are defective but, rather, that they failed to complete the infection.

Table 2.1 Particle-to-PFU ratios of some animal viruses

Virus	Particle/PFU ratio
Papillomaviridae
Papillomavirus	10,000
Picornaviridae
Poliovirus	30–1,000
Herpesviridae
Herpes simplex virus	50–200
Polyomaviridae
Polyomavirus	38–50
Simian virus 40	100–200
Adenoviridae	20–100
Poxviridae	1–100
Orthomyxoviridae
Influenza virus	20–50
Reoviridae
Reovirus	10
Alphaviridae
Semliki Forest virus	1–2

Measurement of Virus Particles

      Although the numbers of virus particles and infectious units are often not equal, assays for particle number are frequently used to approximate the number of infectious particles present in a sample. For example, assuming that the ratio of infectious units to physical particles is constant, the concentration of viral DNA or protein can be used to estimate the number of infectious particles. Biochemical